Download Protein Expression: One By One

NEWS AND VIEWS
specifying and propagating CENP-A localization and centromere identity remain elusive.
Foltz et al. make the intriguing observation
that the nucleolar protein, nucleophosmin-1,
is associated with CENP-A chromatin. This
is interesting in light of previous observations
that centromeres are clustered around nucleoli during interphase in fly and human cells,
that centromere proteins are present in purified nucleoli14 and that a nucleolar transcription factor interacts with CENP-C15. Previous
studies demonstrated that enhancer-blocking
insulators are also associated with nucleophosmin16. It is possible that nucleolus ‘anchoring’ is
a conserved mechanism for sequestering centromeres and other specialized chromatin sites
(Fig. 1). This interaction may be important for
centromeric chromatin assembly, higher order
structure, or promoting or reducing accessibility to other factors. Additional work is needed
to clarify the relationship between nucleoli and
centromeres. Determining how important these
observations are requires direct assessment of
the effects of depleting nucleolar proteins on
the composition and function of centromeric
chromatin, and subsequent kinetochore formation during mitosis.
These studies have identified a large number
of new proteins associated with vertebrate centromeric chromatin, and demonstrated their
importance to CENP-A incorporation, kinetochore formation and chromosome segregation.
Future studies based on these results, and other
intriguing observations, are likely to generate
a more complete understanding of the spatial
organization and functions of these complexes,
as well as the molecular mechanisms involved
in centromere identity, propagation and kinetochore assembly. However, these studies
do bring us perilously close to the end of the
alphabet, should future studies identify additional CENPs.
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Protein expression:
one by one
Most of what we assume to be true about gene expression is based on
genetic and biochemical studies on total pools of molecules and cells,
and even single-cell measurements have so far lacked the sensitivity
to allow observation of protein expression at the single-molecule
level. Now Sunney Xie and colleagues describe two powerful techniques that can track single protein expression, even of low-copy
number proteins.
The technique reported in Nature (440, 358–362; 2006) is based on
the ‘veteran’ gene reporter β-galactosidase (β-gal) that is expressed
from the lacZ gene. Although β-gal is a highly-sensitive probe, the
fluorescent molecules it produces, following substrate hydrolysis, are
not retained in the cell. The authors used closed microfluidic chambers
to trap the fluorescent molecules excreted by the cells in the small
volume of the chambers. In doing so, they were able to obtain realtime quantitative information on gene expression in live Escherichia
coli cells with single molecule sensitivity. Furthermore, they showed
that this technique was also applicable to budding yeast and mouse
embryonic stem cells expressing β-gal from the GAL1 or ROSA promoters, respectively.
The second technique, reported in Science (311, 1600–1603; 2006),
replaces the native lacZ gene with a fusion protein of a fluorescent
tag (YFP–Venus) and Tsr (a membrane protein), so it can be used as
a reporter for monitoring protein expression from the lac promoter.
By tracking the disappearance of the fluorescence signal after photobleaching, the authors could show that each fluorescent peak corresponded to a single molecule.
An overlay of the DIC and fluorescence images of E. coli cells expressing
the fluorescent protein Venus, tethered to the membrane protein Tsr.
Single Tsr–Venus fusion molecules (yellow spots) can be detected when
they anchor to the inner membrane of the cell.
In both studies, the authors concluded that protein molecules are
produced in bursts randomly occurring over time, that the number
of molecules per burst follows an exponential distribution, and that
each burst results from a stochastically transcribed single mRNA.
Furthermore, the burst size and frequency could be determined either
by real-time quantitative monitoring of protein production or by measuring the steady-state distribution of the number of protein copies
within a population of cells.
Xie and colleagues have developed two highly related methods that
allow single-molecule sensitivity at a single-cell level. These techniques
offer new possibilities for understanding gene expression and will allow
genome-wide characterization of low-copy number proteins.
NATURE CELL BIOLOGY VOLUME 8 | NUMBER 5 | MAY 2006
©2006 Nature Publishing Group
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